Recent work has shown that the intracellular environment is organized not only through membrane-bound organelles but also through fluid droplets that emerge through liquid–liquid phase separation (LLPS). Intracellular LLPS has attracted recent attention because these fluid droplets, termed biomolecular condensates or membraneless organelles, seem to play important roles in cells' responses to stress, gene regulation, and pathologies. Our understanding of intracellular LLPS has advanced through many quantitative biophysical techniques. Here, we describe a set of undergraduate lab activities that highlight these biophysical techniques. We use various optical microscopy methods and quantitative image analysis to characterize the physical properties of a model aqueous system that exhibits liquid–liquid phase separation. These lab activities can form a multiweek module that exposes students to this exciting new and interdisciplinary field that investigates how phase transitions organize the cell interior.
Post brain colonization, cancer cells may adhere to and spread along the abluminal surface of the vasculature providing advantageous access to oxygen, nutrients, and vessel-derived paracrine factors. For example, brain-tropic melanoma cells are well-known to invade along or within blood vessels at the invasive front, but the molecular mechanisms that guide this process are not well-characterized. We have used melanoma cells of different phenotypic states (melanocytic versus mesenchymal) to characterize different modes of perivascular invasion in the brain. We find that Sox9hi mesenchymal state melanoma cells undergo pericyte-like spreading along brain blood vessels via a Snail1-dependent process; in contrast, Sox10hi melanocytic state melanoma form proliferative, perivascular clusters. Snai1 deletion in mesenchymal state melanoma cells diminishes Tgfβ-induced expression of Pdgfrβ which impairs Tgfβ/Pdgf ligand driven motility along the brain microvasculature and dramatically reduces perivascular dispersal of melanoma cells throughout the brain post-colonization. These data suggest that, depending on their transcriptional/phenotypic state, some melanoma cells may appropriate signals that typically mediate endothelial cell:pericyte cross talk as an adaptive mechanism for maintaining vessel proximity and motility in the brain microenvironment.
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